Firstly, the PLA-PCL-TPGS random copolymer was synthesized and characterized. Thiolated chitosan greatly increases its mucoadhesiveness and permeation properties, thus increasing the chances of nanoparticle uptake by the gastrointestinal mucosa and improving drug absorption. The PLA-PCL-TPGS nanoparticles were found by FESEM that they are of spherical shape and around 200 nm in diameter. The surface charge of PLA-PCL-TPGS nanoparticles was reversed from anionic to cationic after thiolated chitosan modification. The thiolated chitosan-modified PLA-PCL-TPGS nanoparticles have significantly higher level of the cell uptake than that of thiolated chitosan-modified PLGA nanoparticles and unmodified PLA-PCL-TPGS nanoparticles. In vitro cell viability studies showed advantages of the thiolated chitosan-modified PLA-PCL-TPGS nanoparticles over Taxol® in terms of cytotoxicity against A549 cells. It seems that the mucoadhesive nanoparticles can increase paclitaxel transport by opening tight junctions and bypassing the efflux pump of P-glycoprotein. In conclusion, PLA-PCL-TPGS nanoparticles modified by thiolated chitosan could enhance the cellular uptake and cytotoxicity, which revealed a potential application for oral chemotherapy of lung cancer.Oral chemotherapy is a key step towards ‘chemotherapy at home’, a dream of cancer patients, which will radically change the clinical practice of chemotherapy and greatly improve the quality of life of the patients. In this research, three types of nanoparticle formulation from commercial PCL and self-synthesized  Oral chemotherapy is quickly emerging as an appealing option for cancer patients because it is less stressful, being that the patient will have less hospital visits and can still maintain a close relationship with health care professionals . These fd-α-tocopheryl polyethylene glycol 1000 succinate  is a water-soluble derivative of naturally sourced vitamin E, which is formed by esterification of vitamin E succinate with polyethylene glycol 1000. Previous studies revealed that TPGS was able to improve drug permeability across biological membranes by inhibition of P-gp pumps and, thus, increase the drug absorption capability and decrease P-gp-mediated MDR in cancer cells [Chitosan, a linear polysaccharide derived from chitin obtained from crustacean shells, has emerged as a useful drug delivery matrix because of it polycationic nature, biodegradability, biocompatibility, mucoadhesiveness, and ease of physical and chemical modification . The inter cells -25. In aer cells . The super cells -28. A feer cells . Furtherer cells . Thus, wer cells .d,l-lactide  and d-α-tocopheryl polyethylene glycol 1000 succinate (C33O5H54 (CH2CH2O)23) were purchased from Sigma-Aldrich . ε-caprolactone (CL) were obtained from Acros Organics . Thiolated chitosan (Mw 33000 Da) was from NanoMed Biotech Co. Ltd . Poly(ε-caprolactone) (PCL) (MW 42000 Da), and stannous octoate (Sn(OOCC7H15)2) were also purchased from Sigma . Paclitaxel powder of purity 99.9% was from BioOne Biotech Co. Ltd . Fetal bovine serum was received from Gibco . Methanol and acetonitrile were obtained from EM Science . Deionized (DI) water produced by Millipore Water Systems  was utilized throughout all experiments.ε-caprolactone, lactide, and TPGS in the presence of stannous octoate as a catalyst via ring opening polymerization. In short, pre-weighted amounts of ε-caprolactone, lactide, TPGS, and one drop of stannous octoate were added in a flask. The mixture was heated to 145°C and allowed to react for approximately 16 h. Synthesis was carried out under an oxygen- and moisture-free environment. The product was dissolved in dichloromethane (DCM) and then precipitated in excess cold methanol to remove unreacted monomers and TPGS. The final product was collected by filtration and dried under vacuum. The TPGS content and number-averaged molecular weight of the copolymer was determined by 1H NMR in CDCl3 at 300 Hz .PLA-PCL-TPGS random copolymers were synthesized from w/v) TPGS solution under mild stirring. The mixture was then sonicated for 90 s at 30 W output to form water-in-oil emulsion. The emulsion was further evaporated under ambient conditions overnight to remove DCM. The nanoparticles were harvested by centrifugation at 80,000×g for 15 min and then washed three times to remove the emulsifiers and unentrapped drug. The resulting nanoparticles were finally resuspended in 5 ml of deionized water and lyophilized. The PLA-PCL-TPGS nanoparticles was further modified by thiolated chitosan using a method described previously [g for 15 min. The coumarin-6-loaded nanoparticles were prepared by encapsulation of 0.1% (w/v) coumarin-6 instead of paclitaxel. Thiolated chitosan-modified PCL nanoparticles were fabricated by the same procedure.Nanoparticles were prepared by a modified solvent extraction/evaporation technique ,30. In beviously . PreweigParticle size and size distribution of nanoparticles were measured using dynamic light scattering on a Malvern Zetasizer Nano-ZS90 . The lyophilized nanoparticles were diluted with DI water before measurement. Surface charge of the nanoparticles was determined by laser Doppler anemometry using a Zetasizer Nano Series . All measurements were done in triplicate.The morphology of nanoparticles was characterized by field emission scanning electron microscopy  at 5.0 kV electron high tension. To prepare samples for the FESEM observations, a drop of the particle suspension was placed on a grid or a stud, and the supernatant liquid was removed with a capillary after the particles were allowed to settle. The particles were then coated with platinum layer for 30 s.v/v). Methylene chloride was evaporated under a nitrogen stream until a clear solution obtained. The samples were then used for high-performance liquid chromatography (HPLC) analysis. The column effluent was monitored at 227 nm with a UV–vis detector. The standard size HPLC column (4.6 × 250 mm) is run at a flow rate of 1 mL/min. The drug encapsulation efficiency was defined as the percentage of the drug loaded in the final product. All these experiments were done in triplicates.The encapsulation efficiency (EE) and the actual drug loading of the nanoparticles were measured by high-performance liquid chromatography  as described before ,32. In sw/v Tween 80). The use of Tween 80 in the release media was able to increase the solubility of drug in the PBS and avoided the binding of drug to the tube wall. The nanoparticle suspension was transferred into a dialysis tubing membrane which is sealed at one end with a clamp. The sealed dialysis bag was placed into a centrifuge tube and immersed in 15-ml release medium. The centrifuge tube was placed in an orbital water bath shaking at 130 rpm at 37.0°C. A 10 ml aliquots of samples was periodically removed for HPLC analysis and replaced with fresh medium. The samples were extracted with 2 ml methylene chloride and reconstituted in 5 ml mobile phase. Methylene chloride was evaporated under a nitrogen stream until a clear solution was obtained. The analysis procedure was the same as for the determination of drug EE.Accurately weighted aliquots of drug-loaded nanoparticles (15 mg) were suspended in 5 ml release medium  supplemented with 100 μg/ml streptomycin and 20% fetal bovine serum, was freshened once every 3 days. After reaching 70% to 90% confluence, the cells were harvested with 0.25% of trypsin-EDTA solution  and cultured in 96-well black plate  at the density of 1.3 × 104 cells per well; when the cells reached confluence, the cells were equilibrated with HBSS buffer at 37°C for 60 min and then incubated with coumarin-6-loaded nanoparticle suspension medium. The nanoparticles were well-dispersed in the culture medium at concentrations of 100, 250, and 500 μg/ml. Nanoparticle dispersions were incubated at 37°C in a 5% CO2 atmosphere for 2 h. After incubation with the corresponding nanoparticles, the suspension was removed from the wells, and the cell monolayers were rinsed three times with 50 μl cold PBS (pH 7.4) to remove any traces of nanoparticles left in the wells. After that, the cells were lysed with 50 μl of 0.5% (w/v) Triton-X 100 in 0.2 N NaOH solution . The fluorescence intensity presented in each well was then measured on a GENios Lueifcrase microplate reader  with excitation wavelength at 430 nm and emission wavelength at 485 nm. Cellular uptake efficiency was expressed as the percentage of cell-associated fluorescence vs. that present in the positive control. Culture of human lung cancer cell lines A549 cells and their uptake of the coumarin-6-loaded nanoparticles were performed using the same procedure.Caco-2 cells which were obtained from the American Type Culture Collection  were used in this research to simulate the gastrointestinal barrier for oral chemotherapy. The cells were grown in tissue culture flasks maintained at 37°C in a humidified, 5% COCaco-2 cells were reseeded in the Lab-Tek chambered cover glass system . After the cells were incubated with 250 μg/ml coumarin-6-loaded thiolated chitosan-modified PLA-PCL-TPGS particle suspension at 37°C for 2 h, the cells were rinsed with cold PBS buffer for three times and then fixed with 70% ethanol solution for 20 min. The cells were further rinsed twice with PBS and then counter-stained with 4′,6-diamidino-2-phenylindole dihydrochloride  for the visualization of the cell nuclei. The cell monolayer was rinsed twice with PBS solution and mounted using the Dako fluorescent mounting medium  to be observed by confocal laser scanning microscope . The images of the cells were determined with differential interference contrast channel, and the images of coumarin-6-loaded nanoparticles and the nuclei of the cells stained by DAPI were recorded with the following channels: blue channel (DAPI) with excitation at 340 nm and green channel (coumarin-6) with excitation at 488 nm.4 cells per well and incubated overnight to allow cell attachment. The cells were incubated with drug-loaded PLA-PCL-TPGS nanoparticle suspension, thiolated chitosan-modified PLA-PCL-TPGS nanoparticles, and Taxol®  at 0.25, 2.5, 12.5, and 25 μg/ml equivalent paclitaxel concentrations and blank thiolated chitosan-modified PLA-PCL-TPGS nanoparticles with the same amount of nanoparticles for 24, 48, and 72 h, respectively. At the determined time, the formulations were replaced with fresh DMEM containing MTT (5 mg/ml), and the cells were then incubated for additional 4 h. MTT-containing medium was aspirated off, and 150 ml of DMSO was added to dissolve the formazan crystal formed by living cells. The absorbance at 570 nm was measured by a microplate reader . Untreated cells were taken as a control with 100% viability, and cells without the addition of MTT were used as blank to calibrate the spectrophotometer to zero absorbance. IC50 values (concentration required to reduce cells viability by 50% as compared to the control cells) for each sample was calculated by curve fitting of the cell viability data. The results are expressed as mean ± SD of one representative experiment performed in triplicate, and the experiments were performed three times.A549 cells were counted and seeded in 96-well plates at a density of 0.5 × 10The everted sac method was chosen for the measurement of transportation of paclitaxel across the intestine barrier. It was carried out according to the slightly modified method that was described previously , as foll2, 5% CO2, and contained the test sample in Krebs-Ringer bicarbonate solution at 37°C. The test samples used include: (1) paclitaxel (1 mg) as Taxol®, and (2) thiolated chitosan-modified PLA-PCL-TPGS nanoparticles . In absorption studies, an O2 and CO2 mixture was bubbled into the intestinal mucosa to obtain intestinal peristaltic movement. At certain periods of time, 0.5-mL samples were drawn from inside the intestine and replaced with the same volume of fresh medium. The amount of transported paclitaxel in the samples was measured by the HPLC method.First, a section of about 5 cm of the jejunum was removed from a male rat under ketamine (50 mg/kg) and chlorpromazine (10 mg/kg) anesthesia and washed with Krebs-Ringer bicarbonate solution of pH = 7.4. This section was then gently inverted with a glass rod, and a tube was inserted in one side of the section and tied securely with tape. The other side of the intestine was tied, and 1 mL Krebs-Ringer bicarbonate solution was poured through the hypodermic needle in the tube. The gut sac was placed in a medium saturated with 95% Ot test was used to assess the differences between means. A P value <0.05 was considered to indicate a significant difference.Data were presented as the mean ± SD. Student's Synthesis and characterization of PLA-PCL-TPGS random copolymer.1H NMR in CDCl3. Figure 1H NMR spectroscopy of the PLA-PCL-TPGS copolymer. The signals at 5.2 and 1.69 ppm (peaks a and e) were assigned to the CH protons and methyl protons -CH3 of PLA segment, respectively. The peak at 3.65 ppm (peak c) was assigned to the -CH2 protons of PEO part of TPGS. The lower peaks in the aliphatic region belong to various moieties of vitamin E tails. The peaks at 4.06 (peak b), 2.31 (peak d), 1.60 to 1.70 (peak e), and 1.35 to 1.43 (peak f) were assigned to -OCH2, -COCH2, -CH2 (4 H), and -CH2 (2 H) segments of PCL, respectively [ε-caprolactone, lactide, and TPGS molecular mass were 75%, 15%, and 10%, respectively. However, the ratios of ε-caprolactone, lactide, and TPGS molecular mass which were integrated into the PLA-PCL-TPGS copolymers were 87.18%, 8.17%, and 4.64%.The structure of the synthesized PLA-PCL-TPGS copolymer was detected by ectively . The molThe particle size data of the 5% thiolated chitosan-modified PCL nanoparticles (CNP), unmodified PLA-PCL-TPGS nanoparticles (UNP), 5% thiolated chitosan-modified PLA-PCL-TPGS nanoparticles (TNP), and 20% thiolated chitosan-modified PLA-PCL-TPGS nanoparticles (DNP) fabricated in this research are presented in Table Regarding the drug EE, it can be seen from Table Surface morphology of the 5% thiolated chitosan-modified PLA-PCL-TPGS nanoparticles was inspected by FESEM. Figure in vitro drug release profiles of the CNP, UNP, and TNP in the first 32 days are presented in Figure The in vitro therapeutic effect of paclitaxel in the various formulations for lung cancer treatment [Caco-2 colonic cell line is a widely accepted model to predict the permeability and absorption of compounds in humans . Paclitareatment ,40. The It can be obtained from Figure Figure Figure n = 6). It can be concluded from Figure p < 0.05, n = 6) and 18.72%  for TNP after 48- and 72-h incubation at 10 μg/ml drug concentration. Such advantages of the nanoparticle formulations may be due to the effects of thiolated chitosan and TPGS component of the nanoparticles in enhancing cellular uptake of the nanoparticles. The advantages in cancer cell viability of the TNP > UNP > the Taxol® formulation is dependent on the incubation time. This may be due to the controlled release manner of the nanoparticle formulation. The advantages in cancer cell viability of the TNP > UNP > the Taxol® formulation is also dependent on the drug concentration. The higher the drug concentration, the more significant effects would be obtained.Figure 50, which can be determined by constructing a dose–response curve. Table 50 values of A549 cells after 24-, 48-, 72-h incubations with paclitaxel formulated in CNP, UNP, TNP, and Taxol®, respectively, which are obtained from Figure 50 values for A549 cells were reduced from 2.609, 1.645, and 0.910 to 0.201, 0.122, and 0.106 μg/ml for TNP after 24, 48 and 72 h, respectively. As time goes on, the TNP showed better IC50 values and better in vitro therapeutic effects for A549 cells than commercial Taxol®. This is because the cumulative release of paclitaxel was only 22.63%, 26.52%, and 32.45% for TNP after 24, 48 and 72 h (Figure The advantages in cytotoxicity of the TNP > UNP > Taxol® can be quantitatively analyzed by ICIn this study, we used the everted intestinal sac method for measuring the transporting of paclitaxel from the intestinal barrier. Figure In vitro cell viability studies showed advantages of the thiolated chitosan-modified PLA-PCL-TPGS nanoparticles over commercial Taxol® in terms of cytotoxicity against A549 cells. It seems that the mucoadhesive nanoparticles can increase paclitaxel transport by opening tight junctions and bypassing the efflux pump of P-gp. In short, oral chemotherapy by thiolated chitosan-modified PLA-PCL-TPGS nanoparticle formulation is an attractive alternative approach to the treatment of lung cancer.Three types of nanoparticles were developed from biodegradable self-synthesized PLA-PCL-TPGS random copolymer and commercial PCL for oral delivery of antitumor agents with paclitaxel employed as a model drug, including CNP, UNP, and TNP. The design of the nanoparticle matrix material was made to take full advantages of TPGS in nanoparticle fabrication process such as high emulsification effects and high encapsulation efficiency, as well as improvement of therapeutic effects such as the reduction of P-gp-mediated MDR and superior antitumor efficacy. Thiolated chitosan could greatly increase its mucoadhesiveness and permeation properties, thus increasing the chances of nanoparticle uptake by the gastrointestinal mucosa and improving drug absorption. The data showed that the thiolated chitsoan-modified PLA-PCL-TPGS nanoparticles have significantly higher level of the cell uptake than that of thiolated chitosan-modified PCL nanoparticles and unmodified PLA-PCL-TPGS nanoparticles. The authors declare that they have no competing interests.ex vivo studies and participated in the design of the study. QZ conceived the study and participated in its design and coordination. All authors read and approved the final manuscript.LJ carried out the polymer synthesis, nanoparticle preparation, and cell studies. XL carried out the polymer and nanoparticle characterizations. LL carried out the LJ, XL, LL, QZ are Ph.D., assistant professor, associate professor, and professor, respectively. All authors are from Tianjin Key Laboratory of Biomaterial Research, Institute of Biomedical Engineering, Peking Union Medical College & Chinese Academy of Medical Sciences.


The present study addressed the lack of data on the effect of different types of stretching on diurnal variations in vertical jump height - i.e., squat-jump (SJ) and countermovement-jump (CMJ). We hypothesized that dynamic stretching could affect the diurnal variations of jump height by producing a greater increase in short-term maximal performance in the morning than the evening through increasing core temperature at this time-of-day.Twenty male soccer players  completed the SJ and CMJ tests either after static stretching, dynamic stretching or no-stretching protocols at two times of day, 07:00 h and 17:00 h, with a minimum of 48 hours between testing sessions. One minute after warming-up for 5 minutes by light jogging and performing one of the three stretching protocols  for 8 minutes, each subject completed the SJ and CMJ tests. Jumping heights were recorded and analyzed using a two-way analysis of variance with repeated measures (3 [stretching]×2 [time-of-day]).vs. 4.4±2.64%, p<0.05 for SJ and 10.61±5.49% vs. 6.03±3.14%, p<0.05 for CMJ). However, no significant effect of static stretching on the diurnal variations of SJ and CMJ heights was observed.The SJ and CMJ heights were significantly higher at 17:00 than 07:00 h (p<0.01) after the no-stretching protocol. These daily variations disappeared  to 1.81±4.39% (not-significant) for SJ and from 3.99±3.43% (p<0.01) to 1.51±3.83% (not-significant) for CMJ) after dynamic stretching due to greater increases in SJ and CMJ heights in the morning than the evening (8.4±6.36% Dynamic stretching affects the typical diurnal variations of SJ and CMJ and helps to counteract the lower morning values in vertical jump height. Stretching is considered an essential component of an athlete’s warm-up Maximal short-term performance in vertical jump fluctuates with time-of-day, with morning nadirs and afternoon maximum values in adults The question then arises if dynamic or static stretching produces similar increases in body temperature and vertical jump performance in the morning and the evening. Indeed, the increase in body and muscle temperatures may increase nerve conduction velocity, enzyme activity and muscle compliance The present study addressed the lack of data on the effect of different types of stretching (dynamic and static stretching) on diurnal variations of vertical jump height. We hypothesized that dynamic stretching could affect the diurnal variation of short-term maximal performance by a greater increase in jump height in the morning than the evening through increasing core temperature at this time-of-day.Twenty male soccer players  volunteered to participate in this study. They were affiliated with a professional club competing in the first division of the Tunisian soccer league who trained five times per week for 1.5–2 hours per session. The subjects were informed in detail about the experimental procedures and the possible risks and benefits of the project, and written informed consent was obtained from each athlete or from their parents prior to participation. Subjects were informed that they could resign from participation at any time during the study. The study was conducted according to the Declaration of Helsinki and the protocol was fully approved by the Ethic Committee of the National Center of Medicine and Sciences in Sport of Tunis (CNMSS) before the commencement of the assessments. To have a group without ‘‘extreme types’’, the subjects chosen to participate in the study were all categorized as “moderately morning type” on the basis of their answers to the Horne & Östberg’s One week before the first testing session, participants were familiarized with the vertical jumping and stretching techniques during two orientation sessions. The participants performed six experimental test sessions, with a minimum of 48 hours between testing sessions, in a random order: three in the morning (07:00 h) and three in the evening (17:00 h) with static stretching, dynamic stretching or no-stretching. One minute after completing the specific warm-up protocol (see below), the athletes performed 3 SJs and 3 CMJs with a 30 second passive recovery between each jump. The best performance  was used for further analysis. The experimental procedure is summarized in Oral temperature was recorded by digital clinical thermometer  at the beginning of each test session after the subjects had been in a supine position for 15 minutes and following each warm-up protocol. Core temperature is considered to be a chronobiological marker, i.e., representative of the functioning in the circadian system ad libitum.To minimize confounding factors, instructions related to sleep and diet were given to the subjects before the experiment, as recommended by Bougard et al. Subjects performed a 5 minute self-paced general warm-up consisting of low-intensity aerobic exercise followed by 8 minutes of designated stretching (specific warm-up) protocol for the static stretching and dynamic stretching test sessions or 8 minutes of rest for the no-stretching control condition. The time between finishing the general warm-up and beginning the stretching protocol was approximately 1 minute.The static stretching and the dynamic stretching consisted of 3 lower-extremity exercises that were performed for 3 repetitions for 20 seconds with 7–8 second muscle release between each repetition. The muscles stretched were the hamstrings, the quadriceps and the calf muscles. For the static stretching , each stParticipants were asked to perform maximal vertical SJs and CMJs on an infrared jump system  interfaced with a microcomputer. The Optojump system measures flight and contact times with an accuracy of 1/1000 of a second. For the SJs, subjects got into a squat position and, after a brief pause, jumped upwards as quickly and as high as possible. No downwards motion was allowed immediately prior to jumping upwards. For the CMJs, subjects initiated the jump from an extended leg position, descended to a squat position, and then immediately performed an explosive concentric action to reach maximal height. In both tests, participants were instructed to keep their hands on the hips and to minimize lateral and horizontal displacements throughout the jumps W-test, parametric tests were used. SJ and CMJ heights were analyzed using a two-way analysis of variance (ANOVA) with repeated measures (3 [stretching]×2 [time-of-day]). The difference between the morning and the evening values  during the static stretching, dynamic stretching and no-stretching conditions was calculated and analyzed using a one-way ANOVA with repeated measures. Also, the delta-changes of performance between no-stretching and static stretching, and between no-stretching and dynamic stretching, were calculated at 07:00 and 17:00 h and analyzed using a two-way ANOVA with repeated measures (2 [stretching]×2 [time-of-day]). Oral temperature data were analyzed using a three-way ANOVA with repeated measures (3 [stretching]×2 [before/after]×2 [time-of-day]). When appropriate, significant differences between means were assessed using the Tukey post-hoc test. To assess the data practical significance, effect sizes were calculated as partial eta-squared, ηp2. Test-retest reliability was assessed by intra-class correlation coefficients (ICCs) and standard error of measurement (SEM). Statistical significance for all analyses was set at p≤0.05.Statistical tests were processed using the STATISTICA Software . Mean, and standard deviation (SD) were calculated for the selected variables. Once the assumption of normality had been confirmed by the Shapiro-Wilk The SJ and CMJ heights measured at 07:00 and 17:00 h after static stretching, dynamic stretching and no-stretching protocols are displayed in The SJ showed a high reliability between test-retest sessions (ICC higher than 0.88 and absolute SEM lower than 1.5 cm).p2 = 0.77) and time-of-day . Likewise, the interaction stretching×time-of-day was significant .There was a significant main effect for stretching  with an amplitude of 4.2±2.81%. These diurnal variations were blunted after the dynamic stretching by an attenuated morning-evening difference (4.2±2.81% (p<0.01) vs. 1.81±4.39% (not-significant)) but persisted after the static stretching.After the no-stretching protocol, the vs. 4.4±2.64%, p<0.05). However, the delta-change was not significantly different between the morning and the evening after static stretching.Moreover, SJ height increased after dynamic stretching  and decreased after static stretching  in comparison with no-stretching at 07:00 and 17:00 h. The improvement of performance after dynamic stretching compared to no-stretching was significantly higher in the morning than the evening (8.4±6.36% The CMJ showed a high reliability between test-retest sessions (ICC higher than 0.94 and absolute SEM lower than 1.2 cm).p2 = 0.81) and time-of-day . Likewise, the interaction stretching×time-of-day was significant .There was a significant main effect for stretching  during the no-stretching test sessions with an amplitude of 3.99±3.43%. These diurnal fluctuations persisted during the static stretching sessions but disappeared during the dynamic stretching sessions with a reduced diurnal amplitude (3.99±3.43% (p<0.01) vs. 1.51±3.83% (not-significant)).The vs. 6.03±3.14%, p<0.05). However, jump heights decreased after static stretching in comparison with no-stretching at 07:00 and 17:00 h (p<0.01 and p<0.05 respectively). The delta-change was not significantly different between the morning and the evening after static stretching.Furthermore, in the morning and the evening, jump height during the CMJ increased after dynamic stretching  in comparison with no-stretching, with a greater improvement at 07:00 h than 17:00 h  due to a higher increase in oral temperature in the morning than in the evening . Moreover, compared to before the stretching protocols, the increase in oral temperature after dynamic stretching was significantly higher than after no-stretching and static stretching protocols (both p<0.01).Oral temperatures recorded before and after static stretching, dynamic stretching and no-stretching protocols are presented in The purpose of this study was to determine whether acute static stretching and dynamic stretching had any effect on the diurnal fluctuations of vertical jumping performance. The primary finding was that dynamic stretching may reduce the amplitude of the diurnal variations of SJ and CMJ heights by producing a greater increase in short-term maximal performance in the morning compared to the evening. By contrast, static stretching did not affect the diurnal variations in SJ and CMJ heights.During the no-stretching control condition, the results demonstrated that SJ and CMJ heights were higher in the evening than the morning. Diurnal variations have been demonstrated during continuous  Concerning the effects of static stretching, the present results showed that jumping heights during the SJ and CMJ tests were impaired in the morning and in the evening. Although some previous studies had been unable to show any adverse effect of the static stretching Although some previous studies reported that dynamic stretching did not improve short-term explosive performance Limitations of this study include the fact that all participants were male soccer players. Future investigations should focus on determining any modification to diurnal variations of short-term maximal performance in females. Moreover, short-term maximal performance was tested using only the SJ and CMJ. Therefore, acute effects of stretching on the daily fluctuations of upper body performance should also be investigated. Since the 3 CMJ repetitions were performed after the 3 SJs over a time span of only about 3 minutes, the conclusions from the present study are to be interpreted as an effect not only upon single short-term explosive power efforts but also, to some extent, upon powerful and repeated short bursts of exercise. For more accurate data regarding this issue, a similar protocol could be performed using repeated sprint ability (RSA) – investigating single power, mean power and fatigue index variables and alterations in these with time-of day and after static and dynamic stretching.The results of the present study confirm that (i) static stretching is detrimental and (ii) dynamic stretching is beneficial and can overcome decreases in jump height in the morning. Thus, athletes required to compete in the morning hours and to produce powerful efforts may be advised to include dynamic stretching exercises in their pre-competition warm-ups.


The aim of this study was to establish and to optimize the preparation technology of whole blood internal quality control (IQC) products for blood transfusion compatibility testing. Several B-type RhD-negative blood samples collected from healthy donors were mixed. Two groups of whole blood IQC products, namely, the preservative solution group (PS group) and the saline group, were prepared. The agglutination intensity of IQC sample red cells and anti-B antibody, IgM anti-A antibody and reverse-typing A cell, IgG anti-D and O-type RhD-positive red cells, as well as free hemoglobin concentration in the supernatant of the two groups were detected. The erythrocytes in both groups were damaged to a certain extent during storage, but no evident (above moderate) hemolysis was observed in the stored sample within 42 days. The red cells remained structurally complete and the reaction activity of IgG anti-D reagent remained generally unchanged (P>0.05). Although the reaction activity oscillation of IgM anti-A reagent was observed, the agglutination intensity varied within an acceptable range of 1+. No difference was observed between the preparation methods of the samples, i.e., between the erythrocyte washed with saline and the one washed with red cell preservative solution (P>0.05). The long shelf life, low variance between tubes and stable antigen-antibody reaction activity of the whole blood IQC products prepared using the proposed method can meet the requirements of blood transfusion compatibility testing. In 1950, Levey and Jennings introduced the statistical control theory of industrial product manufacturing into the clinical laboratory and established the initial clinical examination analysis quality control (QC) . Written informed consent was obtained from all participants. All blood specimens were extracted from healthy donors, whose alanine aminotransferase, hepatitis B surface antigen, hepatitis C virus antibody, human immuno-deficiency virus (HIV) antibody and syphilis antibody all met the donor health examination requirements. The whole blood specimens were collected within 10 days prior to the analysis. No hemolysis or abnormal agglutination was observed following centrifugation. Both irregular antibody screening and the direct antiglobulin test showed negative results.μl IgG anti-D was multiplied, continuously diluted with saline and evaluated with an as-prepared 1% O-type RhD-positive erythrocyte suspension. Micro-column gel anti-globulin cards were then used to determine the concentration of dilution when the last agglutination strength of 2+ appeared.A concentration of 100 Several B-group RhD-negative samples from healthy donors were selected for mixing and centrifuged at 1760 × g for 5 min. The supernatant was again subjected to 1760 × g centrifugation for 5 min to remove precipitates. The fluid was used to prepare a plasma pool. The remainder of the hematocrit-mixed erythrocytes were divided into two groups. One group was washed twice with a preservative , while the other group was washed twice with saline . The two groups of erythrocytes were mixed with a preservative and the prepared plasma pool in a volume proportion of 1:2:3, respectively.Gentamicin sulphate 0.05 mg/ml was always added to IQC products. Commercial IgG anti-D reagent was added to IQC products at the dilution rate of the initially occurring agglutination of 2+. The two groups of modified whole blood samples were placed in hard plastic tubes, with 10 products in each group. The tubes were sealed with a cap and preserved at 4°C, with 1 h exposure at room temperature daily.Five units of 0.5 ml group A, B and O, RhD-positive hematid were randomly selected. Once the groups were mixed, the erythrocytes were washed three times with saline . ABO forward grouping was then implemented on the erythrocytes from types A, B and O, respectively, mixed with anti-A, anti-B and standard serum. When grouping was confirmed, the erythrocytes were diluted with saline to obtain a 1% reverse-grouping erythrocyte reagent. The type-O hematocrit was prepared with low ionic strength solution (LISS) to obtain a ready-to-use 1% erythrocyte suspension. All reagent erythrocytes were prepared on the examination day.Measurements were taken at 0, 35, 42 and 49 days after the preparation of the IQC products, including the items below.A WADiana DG-57  automatic blood matching system, DG Gel ABO-CDE blood type card and DG Gel Coombs card (Grifols), as well as the as-prepared 1% A1 and B reverse-grouping reagent erythrocyte and O-group RhD-negative 1% erythrocyte suspension were used for ABO forward and reverse grouping. The RhD grouping determination and irregular antibody screening of all the IQC products (used to examine the erythrocyte B antigen), plasma IgM anti-A and IgG anti-D reaction activity, as well as all agglutination intensity data, were recorded and scored statistically. The scoring criteria  had six A VITROS 5.1 FS Chemistry System and the auxiliary reagent were used in the evaluation .2O2 was added to the mixtures, which were kept at room temperature for 20 min. The tube samples were examined using Multiskan-Mk3 ELISA and the color comparison was read at 492 nm. The following is the obtained result: FHb (mg/l) = (Aexamining-Aempty) / (Astandard-Aempty) × 100.The Trinder reaction method was adopted. A concentration of 0.05 ml plasma was added to the examining tube, HB working solution was added into the standard tube and 0.05 ml saline was added into the empty tube. A concentration of 2.5 ml chromogen buffer was added into each tube and mixed with the contents. Subsequently, 0.5 ml 0.29 mol/l HHemolysis may occur in the samples during preservation. The hemolysis condition was divided into four grades according to FHb content in the supernatant fluid. The four grades are as follows: Grade 0, no hemolysis (FHb <500 mg/l); Grade 1, light hemolysis (500 mg/l ≤FHb <1000 mg/l); Grade 2, medium hemolysis (1000 mg/l ≤FHb <2000 mg/l); and Grade 3, serious hemolysis. If the condition of any sample reached or went beyond Grade 2, the sample was not included in the experiment.An S-4500N scanning electron microscope  was used to examine the morphology of erythrocytes in the control sample.The supernatant from each tube of IQC product was observed for any colony. Special agglutination and obvious color changes were observed.The data obtained were presented as the mean ± standard deviation (SD). A t-test was used to compare the time data of the two groups. One-way analysis of variance (ANOVA) was used to compare the data on various preservation periods within the same sample. P<0.05 was considered to indicate a statistically significant result.During preservation, no obvious change (P>0.05) in agglutination was observed between the hematocrit of the RBC B antigen and standard anti-B serum in the two groups. In addition, no apparent difference (P>0.05) was observed between the two groups within the same preservation period. Fluctuation (P<0.01) occurred in the hematocrit of the agglutination of the IgM anti-A antibody and reverse grouping RBC. Likewise, no obvious difference (P>0.05) was found between the two groups preserved for an equal length of time. No change (P>0.05) was observed in the agglutination of IgG anti-D antibody and O-group RhD-positive RBC. Finally, no obvious difference (P>0.05) was found in the two groups preserved for the same length of time .+ concentration of the supernatant of both groups continuously declined as the preservation period lengthened. Both groups were preserved for 0 days and the saline group was found to have a higher Na+ concentration than the PS sample (P<0.01). No difference was observed between the two samples when preserved for 35 or 42 days. A longer preservation period resulted in higher supernatant K+ in the samples (P<0.01). Meanwhile, no obvious difference (P>0.05) was observed between the two groups preserved for 0, 35 or 42 days. The supernatant LDH concentration of the two groups increased slightly (P>0.05). The lactic acid concentration in the supernatant of the two groups increased (P<0.01). The FHb content in the supernatant of two groups increased as the preservation period lengthened. When preserved for 49 days, the increment reached its peak (P<0.01). The FHb in some IQC products reached Grade 2 hemolysis, with no obvious difference (P>0.05) between the two groups preserved for 0, 35, 42 and 49 days , injury occurred on RBC to some extent, including an increase in KWhen IQC products were preserved for 49 days, the remaining supernatant in every sample was insufficient. Thus, only one biochemical index, FHb, was tested. The test showed that certain IQC products reached Grade 2 hemolysis. The above findings prove that whole blood IQC products retained good stability within 42 days.The addition of an RBC preservative into whole blood or RBC may effectively extend the shelf life of blood products –19. To pBacteriostasis should also be considered. The initial research showed that whole blood IQC products with added glucose, adenine and mannite were preserved at 4°C for over 35 days. In this research, the washing hematocrit RBC and RBC preservative were mixed with plasma at 1:2:3. The mixed liquid was then placed into hard plastic tubes, each of which contained 6 ml control. Then, 0.05 mg/ml gentamicin sulphate was added.The difference between IQC products prepared using the proposed method and the ordinary test samples was evident in the supernatant (RBC preservative and plasma). The supernatant of the former was more significant than that of the latter. The amount of plasma used for IQC products is significantly greater than that used for hematocrit RBC. This feature of IQC products enables the preservation and increases the utilization efficiency of RBC. Although the addition of preservative into IQC products may dilute the antibody to a certain extent, the effect on the reactive intensity remains within the acceptable range. Moreover, the weakly positive antibody control material caused by dilution contributed to the monitoring of the sensitivity of detection system. Clean containers and a bacteria-free solution were used in the preparation and some antibiotics were added to prevent bacteria from growing. The IQC products in the two groups could be effectively preserved for more than 42 days.In summary, IQC products prepared using the proposed technique have the advantages of long preservation time, low vial-to-vial variation and stable antigen and antibody reaction activity, thus satisfying the requirements of blood transfusion compatibility testing. Such IQC products are suitable for application in the field of blood transfusion compatibility testing.


Type 2 diabetes mellitus (T2DM) is characterized by a progressive failure of pancreatic β-cell function (BCF)with insulin resistance. Once insulin over-secretion can no longer compensate for the degree of insulin resistance, hyperglycemiabecomes clinically significant and deterioration of residual β-cell reserve accelerates. This pathophysiology hasimportant therapeutic implications. Ideally, therapy should address the underlying pathology and should be started earlyalong the spectrum of decreasing glucose tolerance in order to prevent or slow β-cell failure and reverse insulin resistance.The development of an optimal treatment strategy for each patient requires accurate diagnostic tools for evaluating theunderlying state of glucose tolerance. This review focuses on the most widely used methods for measuring BCF within thecontext of insulin resistance and includes examples of their use in prediabetes and T2DM, with an emphasis on the mostrecent therapeutic options (dipeptidyl peptidase-4 inhibitors and glucagon-like peptide-1 receptor agonists). Methods ofBCF measurement include the homeostasis model assessment (HOMA); oral glucose tolerance tests, intravenous glucosetolerance tests (IVGTT), and meal tolerance tests; and the hyperglycemic clamp procedure. To provide a meaningfulevaluation of BCF, it is necessary to interpret all observations within the context of insulin resistance. Therefore, this reviewalso discusses methods utilized to quantitate insulin-dependent glucose metabolism, such as the IVGTT and theeuglycemic-hyperinsulinemic clamp procedures. In addition, an example is presented of a mathematical modeling approachthat can use data from BCF measurements to develop a better understanding of BCF behavior and the overallstatus of glucose tolerance. A relatively low precision has been reported for estimates based on the HOMA model   was based on the calculation of the rate of glucose disappearance (KG) and the supra-basal area under the plasma-concentration time curve (AUC) of insulin concentration (ΔAUCINS) over an observation period of 40 min (T). The value for α was assumed to be equal to the regression line slope between KG/(ΔAUCINS/T) and SI in control participants. The CSI and the SI showed high correlation (r2=0.68-0.96) and regression line slopes of ~1 in the majority of groups. In patients with T2DM, the CSI tended to overestimate the SI. The CSI showed good correlation with the M value (r2=0.82) and an inverse relationship with the body mass index (BMI) associated with the SI. The authors concluded that, in situations of low insulin sensitivity (such as T2DM), the CSI may suffer from inaccuracy, but unfortunately the SI may also exhibit inaccuracy under these conditions. The authors also concluded that the shorter test achieved a good approximation of the results obtained with the usual 3-hour IVGTT with minimal model analysis and the insulin sensitivity values derived from a euglycemic-hyperinsulinemic clamp. The obvious advantages of the shorter method are the time requirements and the lower cost when compared to the original, longer IVGTT procedure.Tura et al.  proposedThe IVGTT is amenable for use in a routine clinical setting with minimal patient risk and enables, perhaps, the most accurate measurement of the first-phase (acute) insulin secretory response to a nutrient stimulus. However, because most offices dealing with patients who have diabetes are not equipped with the materials and personnel qualified to perform and interpret the results, in our opinion this should not be encouraged. Although it is an artificial, non-physiological parameter, mechanistically, this rapid insulin release (~10 min post-bolus) is almost certainly due to membrane-docked secretory granules within β-cells . The IVGsee below) were designed to assess insulin secretory patterns under more physiological conditions than those of the IVGTT, while capturing the complex interrelationship between glucose levels, insulin secretory response, insulin action, and hepatic insulin extraction  and nateglinide) does not share structural characteristics with the SFU drugs, they do have a similar mechanism of action. The assessment of BCF using this method, on a background of "glitinide" therapy, falls short for the same reasons explained above , 67. Due1c in patients with T2DM who are started on therapy with TZD, glycemic control is maintained for longer periods of time, presumably due to the preservation of BCF ), and TZD (ROSI) monotherapies were directly compared in a group of newly diagnosed patients with T2DM over a median period of 4 years . After 5P<0.05 between groups). However, this difference was substantially greater between weeks 4 and 42 (mostly due to an elevated SFU effect) than between weeks 52 and 104 when HOMA-B faded back towards baseline in the SFU group. HOMA-B in the PIO group gradually increased up to week 24, then reached a plateau at a level higher than baseline. The calculated HOMA-S decreased from baseline with SFU to a nadir by week 8 and increased with PIO to plateau by week 24 (P<0.0001 between groups). In a 26-week study of drug-naïve patients with T2DM, therapeutic doses of PIO given as monotherapy increased the IGI during an OGTT   and +9.89±3.19 nmol/L*min [P=0.003], respectively). In addition, VILDA monotherapy increased the acute plasma insulin response to arginine (AIRarg) by +5.0±1.8 nmol/L*min, versus a decrease of -0.8±1.8 nmol/L*min with placebo (P=0.03 between groups). No effect on insulin sensitivity was observed, as the M values were increased by +0.77±1.9 mg*kg−1*min−1 and +0.55±1.72 mg*kg−1*min−1, for VILDA and placebo, respectively. Of interest, there was a trend for a VILDA-induced increase in the calculated DI  by +49.6±15.0 nmol*mg*L-1*kg-1, but this did not reach statistical significance. Unfortunately, all between-group differences had disappeared by the end of the 12-week washout period, suggesting that there was no permanent improvement of BCF associated with VILDA therapy. This type of meager response to DPPI therapy was confirmed in another clinical study in drug-naïve patients with T2DM using SAXA monotherapy versus placebo for 24 weeks, followed by open-label SAXA for an additional 24 weeks [P<0.0001). Furthermore, SAXA monotherapy was accompanied by an increase in postprandial insulin and C-peptide AUC, with suppression of postprandial glucagon AUC, relative to placebo. A small improvement in insulin sensitivity in the postprandial , but not in the fasting (HOMA-IR), state was also demonstrated. However, because the MI and IGI did not change, no clinically meaningful improvement in whole-body insulin resistance can be inferred from these data. At the end of the open-label extension with SAXA 10 mg QD, post-challenge plasma glucose at the 120-min time point during the OGTT had decreased by -3.7 mmol/L from baseline (P<0.05). Total PPG AUC was also significantly reduced (P<0.05). Finally, on a background of TZD (primarily PIO), SAXA for 24 weeks decreased glucose concentration at all time points during an OGTT [P<0.0001). The IGI also increased from baseline with SAXA, but decreased with placebo.The effects of DPPIs on measures of the relationships among whole-body glucose metabolism, tissue insulin resistance, and islet-cell function after a nutrient load can be exemplified by a report from Foley et al. . In drug24 weeks . During  an OGTT . At the et al. [P=0.002) in this placebo-controlled study. According to this model, the slope of the β-cell dose response, the derivative component, and the potentiation factor were not affected.An example of using a comprehensive mathematical modeling approach to understand the physiological changes in drug-naïve patients with T2DM during DPPI treatment was reported by Mari et al. . Four we4) and the review article by Van Genugten et al. [ In summary, improvements in HOMA-B and some dynamic measures of BCF have consistently been demonstrated across the DPPI drug class. However, these improvements were not sustained after drug withdrawal. There are no apparent differences among the DPPI drugs currently available. Further examples can be found in . Insulin sensitivity was also 61% higher in ad libitum-fed exenatide-treated rats than in pair-fed control rats (P<0.003), despite comparable HbA1c, fasting glucose, fasting insulin, total cholesterol, HDL-C, and food consumption. In ad libitum-fed exenatide-treated rats, the index of “β-cell mass x insulin sensitivity” was 63% greater than in the pair-fed control rats (P<0.05). Thus, the study concluded that exenatide infusion increased β-cell mass to a greater extent than would be expected in animals with comparable degrees of insulin resistance. These data suggest that exenatide may exert a direct trophic effect on pancreatic β-cell mass, presumably by stimulating islet neogenesis, independent of any improvements in metabolic parameters. Numerous nonclinical experimental models of T2DM and insulin resistance have been studied in an effort to assess the effects of GLP-1RAs on islet-cell morphology and on BCF, in conjunction with measurements of the degree of insulin resistance , 104. Geet al.  used thein vitro and in vivo experimental animal studies, and definitive data in humans are still lacking. In another example, the demonstration of an increased expression of the transcription factor PDX-1/IDX-1, a factor essential for the development and regeneration of the endocrine pancreas, suggested that this may be one important intracellular mechanism by which GLP-1RAs can enhance β-cell mass. GLP-1RAs can also induce AR42J pancreatic tumor cells, a cell line that is negative for islet hormones and their respective mRNAs, but can proliferate and differentiate into cells with positive immunohistochemical staining for insulin. Nonetheless, a functional correlation of these morphological and developmental findings in islet cells has not yet been established in patients with T2DM.Various reports have also demonstrated augmentation of pancreatic β-cell mass with continued exposure to a GLP-1RA, primarily in animal models of insulin resistance and T2DM. These observations have been interpreted as potential evidence that GLP-1RAs may promote β-cell neogenesis and proliferation, concurrently with suppression of β-cell apoptosis , 104. InP=0.01) [The effects of GLP-1RAs on measures of fasting homeostasis (HOMA) can be exemplified by results obtained in a clinical trial of ExBID monotherapy for 24 weeks in treatment-naïve T2DM patients where HOMA-B improved by 28%, compared with only 6% in the placebo control group, starting with baseline values of 45.3%-50.0% across groups (P=0.01) . In clinP=0.01) -121. In P=0.01) , 117-125P=0.01) , 126. P=0.02) [P=0.001 vs. placebo) [P<0.001 vs. placebo) [P<0.01) [P<0.0001) [P<0.001) on a MET+SFU background therapy [P<0.01 vs. baseline; P<0.05 vs. SFU). In clinical pharmacology studies conducted in T2DM patients, exenatide therapy was shown to modulate serum insulin in a manner dependent upon both the exenatide dose and the ambient glucose concentration -129. ExBP=0.02)  and, in placebo) . In a seplacebo) . Further[P<0.01) , with fu<0.0001) . In a di therapy  and anot therapy . In the 2). Similar results have been reported in clinical trials in which patients with T2DM had a concomitant background therapy that included a TZD as monotherapy or in combination  , 125. InP<0.0001), and the median ratio of proinsulin-to-insulin also decreased versus the placebo group (P≤0.02) [P<0.0001 between groups) in a group of patients with T2DM receiving adjunctive treatment with either MET, SFU, or MET+SFU [P=0.0019) [P<0.0001), whereas the mean ratio of proinsulin-to-insulin versus placebo decreased significantly (P<0.05) [P≤0.02) [P=0.01). LIRA QD at three different doses for 14 weeks improved mean HOMA-B by 75%-134% versus placebo ((P≤0.02) . In anot MET+SFU . In this=0.0019) . When LI(P<0.05) . In cont(P<0.05) . When co[P≤0.02) , though P<0.001) versus monotherapy with either MET (+1.4±0.04), PIO (+1.3±0.05), or SITA (+1.3±0.04) [P<0.05) [In drug-naïve patients with T2DM, the difference between the HOMA-B value at the 26-week endpoint and the baseline HOMA-B value was significantly greater with ExQW monotherapy  . In addi[P<0.05) . Signifi[P<0.05) . 1c to approximately 6.8% [P<0.0001). Arginine-stimulated plasma C-peptide concentrations increased 3.2-fold when compared with baseline after exenatide therapy versus only 1.3-fold with GLARG therapy (P<0.0001 between groups). However, these impressive results were not sustained, and measures of BCF had returned to pretreatment levels in both groups 4 weeks after drug discontinuation. Of interest, in a follow-up to this observation, after 3 years of treatment and 4 weeks off-drug, first-phase insulin secretion adjusted for insulin sensitivity (DI) demonstrated a sustained improvement in the exenatide group, whereas GLARG slightly reduced this measure of BCF (P=0.028 between groups) [M value), although 4 weeks after drug discontinuation insulin sensitivity remained improved in the exenatide group , but not in the GLARG group. The calculated DI measured during the second-phase (10-80 min) insulin secretion during the hyperglycemic clamp was not significantly different between groups.The effects of GLP-1RAs on measures of the dynamic relationship between glucose metabolism and BCF after a nutrient load can be exemplified by reports from Bunck and colleagues , 133. Ately 6.8% . Under a0-10 min) during an IVGTT by 103% compared with placebo (P=0.05), but did not achieve as large a response as was observed in healthy, untreated, non-diabetic volunteers [0-10 min and some, but not all, doses of LIRA also improved the second-phase insulin response (AUC19-40 min) compared with placebo. Similar to the study mentioned above for exenatide [P<0.05) [0-30 min) and in the IGI, which was greater than that observed in the SITA-treated group (P≤0.02). In a similar study using a two-way crossover design, patients with T2DM were randomized to a single subcutaneous injection of LIXI or placebo [4).In an independent and separate 14-week study, LIRA improved first-phase insulin response  . One add[P<0.05) . In this placebo . Two houet al. [P=0.02 between groups). At week 30, the 2-hour post-meal-to-basal potentiating factor ratio was estimated to have increased to 1.53±0.10, compared with only 1.15±0.06 in the placebo group (P=0.01 between groups). Thus, the mathematical model predicted that ExBID treatment would be associated with an upward shift of the β-cell dose response, with an enhanced potentiating of insulin secretion.An example of using a comprehensive mathematical modeling approach to understand the physiological changes during GLP-1RA treatment is provided in a report by Mari et al. . In thisIn summary, because the dynamic tests are more reliable methods of assessing changes in BCF than are the homeostatic tests, it seems reasonable to assume that GLP-1RAs are more likely capable of restoring physiological β-cell insulin secretion patterns in all stages of glucose intolerance, as compared with insulin and other antidiabetes therapies. For the most part, however, any perceived improvements in BCF while patients are in treatment are not sustained upon discontinuation of the drugs. Although these effects may be apparently lost immediately after discontinuation of the GLP-1RA therapy, there seems to be a long-term imprint effect on the first-phase insulin secretion, which is worth pursuing in all patients with T2DM who can tolerate, and are good candidates for, GLP-1RA therapy. Finally, improvements in both homeostatic and dynamic measures of BCF have been consistently demonstrated across the GLP-1RA drug class, but whether these have any impact on the natural history of the disease, which translates into clinical benefits to patients with T2DM, remains to be determined. 6). Based on extensive review of the literature, it appears that the development of an optimal treatment strategy for each patient with T2DM, as well as for those with prediabetes, will require an accurate early diagnostic assessment of the degree of residual BCF within the context of each individual’s insulin resistance. In this review, we focused on the most widely used methods for the evaluation of BCF and their clinical applications.The pathophysiology of T2DM is characterized by progressive β-cell failure on the background of insulin resistance  insulin secretion and second-phase (more prolonged) insulin secretion after an intravenous bolus of glucose. The model assumes that glucose acts to increase its own utilization and to retard its endogenous production in direct proportion to plasma glucose concentration. Insulin is assumed to synergize with these glucose effects and promote further glucose disappearance from plasma in a concentration-dependent manner. The IVGTT is amenable for use in a routine clinical setting with minimal patient risk and yields perhaps the most accurate measurement of the first-phase insulin secretory response to a carbohydrate stimulus. The IVGTT is limited, nonetheless, because it cannot predict the variable glucose absorption rates from the gut, and thus ISRs calculated using this approach represent a theoretical and artificial response to a non-physiological intravenous glucose load. The OGTT and MTT provide means of assessing insulin secretory patterns in more physiological conditions, as these tests include the gastrointestinal incretin effect that follows oral nutrient ingestion. The primary measures of the β-cell pancreatic responses are also the determination of the magnitude of the first- (acute) and second-phase insulin secretion combined, after an oral nutrient load. Typically, these values are captured from the incremental AUC. Although the acute and prolonged phases of insulin secretion using these oral intake methods cannot be as sharply delineated as in the IVGTT, the inclusion of the glucose absorption from the gut is one important factor that contributes to the overall pancreatic insulin secretory process. Use of a two-compartment oral minimal model can provide two global indices of BCF, namely the ISR related to increased adiposity and a β-cell index that takes into account a patient's glucose tolerance state. The MTT is more difficult to standardize and more cumbersome to administer than the OGTT, but it more closely reproduces the normal physiologic state after consumption of a mixed meal, and thus provides data of greater clinical and nutritional relevance.The hyperglycemic clamp is a highly reproducible method of assessing β-cell sensitivity to glucose under maximal stimulatory conditions. This technique enables accurate and reproducible follow-up estimates of BCF in any condition where the insulin response either improves or deteriorates as a result of changes in plasma glucose levels over time. It can be applied to investigate BCF in longitudinal studies of small populations, and to compare BCF before and after therapeutic interventions.i.e., the euglycemic-hyperinsulinemic clamp procedure) was discussed to provide a background for the correct assessment of insulin secretion in patients with prediabetes and T2DM under various therapies. Finally, the Mari/Ferrannini mathematical model represents an approach that derives data from some of the discussed dynamic tests used in evaluating BCF and can be used to develop population-wide conclusions concerning the overall status of BCF and glucose tolerance. The model incorporates most factors known to be involved in the regulation of the pancreatic β-cell response to glucose, allowing for theoretical explorations of the dynamic relationships between glucose and insulin concentrations. The gold-standard method for quantitating the degree of insulin resistance , DPPIs and GLP-1RAs have been demonstrated to augment pancreatic β-cell mass under conditions of insulin resistance or overt T2DM In conclusion, we believe that early and more accurate BCF assessment can be achieved with the use of methods that provide an index of insulin secretion that takes into consideration the degree of insulin resistance in each individual. The DI comes close to this goal, as does the insulin secretion/insulin resistance index. The insulin secretion estimates derived from the C-peptide deconvolution curve may provide similar results. These indices can be derived from dynamic tests such as the OGTT and MTT with indices and from the IVGTT minimal model. To acquire an enhanced and reliable index of insulin resistance, the euglycemic-hyperinsulinemic clamp procedure may be performed in small groups of subjects. These indices are very sensitive and reproducible over time and can be useful tools in the follow-up of β-cell failure following therapy. Slowing β-cell failure is an important part of the overall treatment of diabetes, and any effective therapeutic strategy should be able to slow the progression in the initial stages of the disease (prediabetic state). This is critical because we know that the disease usually begins 5-10 years prior to the actual clinical diagnosis. Applying the HOMA model, and/or a standard OGTT, may be more convenient and relatively simple, but the results are less sensitive for the detection of changes in BCF and should not be used as the only tools for the assessment of pancreatic reserve. The estimation of BCF in the advanced stages of the disease can be attained with the use of routine determination of plasma insulin, C-peptide, and glucose concentration. However, there is a good chance that β-cell failure has reached an irreversible phase and it is too late to act, since the ultimate goal is to achieve a meaningful retardation of the appearance of hyperglycemia and the development of frank diabetes and its complications. Whether better knowledge of the degree and rate of deterioration of BCF early in the disease process provides an additional tool to help manage diabetes and prediabetes remains to be determined.


Well-run screening programs for cervical cancer in the population at risk have been shown to result in a sharp decrease in the incidence and mortality of cervical cancer in a number of large populations. Expression patterns of a recently identified biomarker family, microRNA, appear to be characteristic of tumor type and developmental origin. Several tumors have been reported to actively release exosomes carrying microRNAs. The present study has determined the association of microRNAs with cervical cancer-derived exosomes. The cervical cancer-derived exosomes were enriched in the cervicovaginal lavages specimens and the abundance of exosomes and exosomal microRNAs was detected by electron microscopy, western blot analysis, RT-qPCR and microRNA target reporter vector. The microRNA-21 and microRNA-146a, which were up-regulated in cervical cancer patients, were associated with the high levels of cervical cancer-derived exosomes. In conclusion, we demonstrated the abundance of exosomes in the cervicovaginal lavage specimens of women with cervical cancer. Furthermore, our results indicated that abnormally high levels of microRNA-21 and microRNA-146a existed in the cervical cancer-derived exosomes and the two microRNAs were functional in 293T cells. Despitenvasive) . Indeed,nvasive) ,10.MicroRNAs (miRNAs) are non-coding RNA molecules of approximately 22 nucleotides that regulate gene expression in organisms ranging from nematodes to humans , and in The majority of microRNAs are present within the cell, and only a smaller number of them have been detected outside cells, including various body fluids –27. SurpThe present study is to determine whether the cervical cancer-derived exosomes contain abnormally high levels of microRNA-21 and microRNA-146a, which are up-regulated in cervical cancers, and to evaluate whether the exosomal microRNA-21 and microRNA-146a can potentially serve as useful biomarkers for cervical cancer diagnosis.2.2.1.Cervicovaginal lavages specimens were collected from 45 patients with cervical cancers, 25 HPV-positive subjects and 32 normal HPV-negative subjects. Exosomes were isolated from the specimens by ultracentrifugation. The exosomes were examined and confirmed by electron microscopy. Under electron microscopy, the pellets were spherical in shape, with an average diameters varying between 30 and 110 nm , consist2.2.p < 0.01). The HPV infection seemed to contribute to the up-regulation of microRNA-21 in cervical tissue [p < 0.05). The exosomal miRNA-146a level in cervicovaginal lavage specimens of the cervical cancer patients was also significantly higher than that of the HPV-positive subjects and HPV-negative normal subjects (p < 0.01). However, there was no significance in the miRNA-21 or miRNA-146a level in the supernatant of cervicovaginal lavage among the three groups. In addition, the relative miRNA-21 or miRNA-146a level in exosomes was significantly higher than in the supernatant  is used to screen potentially pre-cancerous and cancerous processes in the endocervical canal, with a sensitivity of about 68% and a specificity of about 75% [In this study, we examined the morphology of exosomes isolated from cervicovaginal lavage specimens of cervical cancer patients by electron microscopy, and measured the expression of exosome-specific markers (CD9 and CD63) by Western blot analysis. The electron microscope images indicated that the exosomes were spherical or cup-shaped, with a diameter of 30–100 nm. The exosome-specific markers, CD9 and CD63, existed primarily in the vesicles collected by ultracentrifugation, rather than the supernatant of the cervicovaginal lavage specimens. The RNA levels of microRNA-21 and microRNA-146a were both elevated in the enriched exosomes from the cervical cancer patients. The exosomal microRNAs were further investigated by using a calcium ionophore (A23187) as well as a microRNA-21 sensor reporter vector. MicroRNA-21 and microRNA-146a released from a cervical adenocarcinoma cell line, Hela, were significantly up-regulated after the stimulation with A23187. The exosomes released by Hela cells reduced the luciferase activity of the microRNA-21 sensor reporter in 293T cells. Taking together, this study reports for the first time the elevated levels of exosomal microRNAs in cervicovaginal lavage specimens of cervical cancer patients, implying a potential application in non-invasive cervical cancer screening. Well-run screening programs for cervical cancer in the population at risk have been shown to result in a sharp decrease in the incidence and mortality of cervical cancer in a number of large populations –51. The bout 75% . The accbout 75% . Newer tbout 75% . The HPVbout 75% . ObviousExpression profiling of microRNAs has been shown to be highly specific for the types and origin of cancers, as well as the progression of diseases. However, it is difficult to detect the microRNA profile of abnormal tissues without invasive procedure, and the microRNA levels from the abnormal tissues were usually not enough to cause changes in the level of circulating microRNAs. Therefore, it may be a feasible approach to detect the abnormal microRNAs in secretions from the primary tumor site, particularly for epithelium-derived cancers, such as cervical cancers, nasopharyngeal carcinomas, lung cancers, gastric cancers, carcinoma of colon, and so on. In this study, we determined the levels of microRNA-21 and microRNA-146a in the supernatant of cervicovaginal lavage specimens after ultracentrifugation, and no statistical significance of the two microRNAs was observed between cervical cancer and normal groups . Surpris4.4.1.Forty-five patients with cervical cancer of stage IB–IIB , 25 normal subjects with HPV-positive (HPV18) and 32 normal subjects with HPV-negative were enrolled in this study. There was no cervical (pre) cancerous disease in all normal subjects (with or without HPV infection), screened by liquid-based cytology. Cervicovaginal lavage fluid from the 45 patients was collected prior to any treatment. The normal control cervicovaginal lavage fluid was collected from volunteers with or without HPV infection. Written informed consent was obtained from all of the subjects for using their cervicovaginal lavage fluid for research purposes. The use of clinical material was approved by the institutional review board according to the guidelines of the Dutch Federation of Medical Research Associations.4.2.Hela cell line was purchased from ATCC and maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  at 37 °C with 5% CO2. Calcium Ionophore A23187  was dissolved in DMSO with a concentration of 0.5 mM, and stored at −20 °C before use. CD9 and CD63 rabbit polyclonal antibodies were purchased from Santa Cruz Biotechnology ; the goat anti-rabbit secondary antibody was purchased from Sino Biological . The Reporter pEZX-MT-01 plasmid with microRNA-21 targets and the pEZX-MT-01 control plasmid were purchased from GeneCopoeia, Inc. .4.3.g for 10 min at 4 °C to remove the cells and debris, and then was centrifuged at 13,500× g for 30 min at 4 °C, followed by ultracentrifugation at 100,000× g for 2 h at 4 °C. The pellet was stored at −80 °C for subsequent applications.PBS (10 mL) was utilized for the vagina and cervix lavage . The lav4.4.The ultracentrifuged pellets from cervicovaginal lavage specimens were resuspended in TBS and applied to a grid covered with collodion, then negatively stained with 2% phosphotungstic acid . The microphotographs were obtained using an HD-2000 scanning transmission electron microscope .4.5.The vesicles were resuspended in cell lysis reagent , and supplemented with protease inhibitor cocktail . After protein concentration determination using the BCA Protein Assay Reagent Kit , equal amounts of protein and sample buffer were separated in a 12% gradient SDS-PAGE gel; the gel was stained with Coomassie blue, or transferred to polyvinylidene fluoride membrane. The blotted membrane was blocked with tris-buffered saline containing 5% milk and incubated with the CD63 or CD9 rabbit polyclonal antibody , followed by incubation with horseradish peroxidase-coupled secondary antibody . The proteins were detected using enhanced chemiluminescence . All immunoblots are representative of at least three independent experiments. ELISA was used to measure the exosome membrane-anchored CD9. The limit of detection was 25 pg/mL. 96 well-plates  were coated with anti-CD63 polyclonal antibody  and blocked by PBS containing 0.5% BSA. Purified exosomes from the cell culture supernatants or from the cervicovaginal lavage specimens were detected by anti-CD9 Mab  according to manufacture instruction, and optical densities were read at 450 nm.4.6.Exosomal miRNAs were isolated using mirVana miRNA Isolation Kit  post the RNAse A  digestion of miRNAs outside exosomes. Reverse transcription (RT) reactions and quantitative polymerase chain reactions (qPCR) were performed using the MicroRNA TaqMan Reverse Transcription Kit and the TaqMan MicroRNA Assays  for mir-21 or mir-146a. Three microliters of cDNA were used as a template in a 15-μL PCR reaction. The template cDNA was amplified using specific primers for microRNA-21 or mocroRNA-146a. PCR reactions for each sample were run in triplicate.4.7.5 293T cells were transfected with 50 nM of miRNA control or miR-21 mimics, and 0.5 μg of pEZX-MT01 reporter plasmid with Lipofectamine 2000 . The medium was replaced with DMEM containing 10% FBS at 6 h post-transfection. Cells were harvested at 24 h post-transfection for analysis. Exosomes induced by A23187  from Hela cells  were isolated as mentioned above, and inoculated 293T cells  with a 3-mL volume. At 2 h post inoculation, cells were transfected with 0.5 μg pEZX-MT01 reporter or control plasmid. Cells were harvested at 24 h after transfection. Luciferase activity was determined by using a Promega luciferase assay system . Experiments were conducted in triplicate, and means were determined.Reporter plasmid with microRNA-21 sensitive sites (pEZX-MT01) was purchased from GeneCopoeia, Inc. . Briefly, 1 × 104.8.Cell viability was determined by methyl thiazolyl tetrazoliym assay (MTT). Hela cells were plated in 96-well plates and grown up to 85%–90% confluency. Hela cells were then treated with 0, 0.25, 0.5, 0.75 or 1 μM A23187 for 0, 12, 24 or 48 h. After that, the medium was replaced with 50 μL MTT solution, and the cells were incubated at 37 °C for 2 h. The MTT solution was then discarded and 150 μL DMSO was added to dissolve the precipitate completely at room temperature. The optical density was measured at 570 nm using a spectrophotometer .4.9.t test were performed to compare the differences between two groups. Correlation of the expression levels of microRNA-21, or microRNA-146a with the level of exosomal CD9 was calculated with the pearson correlation analysis of SPSS 17.0 software . Statistical significance was considered with p < 0.05 and p < 0.01.The results are expressed as mean ± standard deviation. Student 5.In summary, we found abundant exosomes in the cervicovaginal lavage of women with cervical cancer. Abnormally high levels of microRNA-21 and microRNA-146a existed in the cervical cancer-derived exosomes and were functional in 293T cells.


DC-offset and DC-suppression are key parameters in bioelectric amplifiers. However, specific DC analyses are not often explained. Several factors influence the DC-budget: the programmable gain, the programmable cut-off frequencies for high pass filtering and, the low cut-off values and the capacitor blocking issues involved. A new intermediate stage is proposed to address the DC problem entirely. Two implementations were tested. The stage is composed of a programmable gain amplifier (PGA) with DC-rejection and low output offset. Cut-off frequencies are selectable and values from 0.016 to 31.83 Hz were tested, and the capacitor deblocking is embedded in the design. Hence, this PGA delivers most of the required gain with constant low output offset, notwithstanding the gain or cut-off frequency selected. Most ofParasitic DC voltage levels appear at the output of the IA. They are produced by several factors such as impedance imbalance from the input electrodes, electrode contact potentials, input bias currents and conversion from differential mode to common mode ,3. TheseThe objective of this paper is to present a new compact PGA stage to perform most of the key tasks in a bioelectric amplifier with a constrained output DC-level. An IA front end combined with this PGA stage, as displayed in 2.2.1.As shown in o, and the whole stage thus behaves as a high-pass filter (HPF) with gain G.The circuit operation relies on an inverter stage to amplify the input, and a feedback integrator in order to eliminate the output DC level. The integrator acts as a low-pass filter. Hence, when feedback is applied, the DC component is eliminated at the output vThe circuit can be easily solved to obtain the transfer function in B, as low IB op-amps are employed. Otherwise, compensation resistors could be required at the op-amp inputs. A bioelectric amplifier demands gain and HPF selection to accommodate to the bioelectric signal. The configuration in 2 among a set of predefined values. Likewise, a set of HPF cut-off frequencies can be obtained by switching on R3 or C. However, low cut-off frequencies require high values of R3 or C. Standard top resistor values are constrained to megohms, and large capacitors, usually electrolytic, have remarkable leakage currents that act as a resistance in parallel with C. This effect is transferred to a DC level at the stage output, vo, which depends on the leakage current. Hence, the achieved outcome during the tests was that switching on electrolytic capacitors with different leakages gave an undesired set of different output DC levels at vo.These formulae ignore the op-amp input bias currents I2.2.The objective is to obtain a programmable gain amplifier (PGA) with HPF selection. Output DC offset must be low and independent from the selected cut-off frequency. Hence, two new configurations were implemented, as shown in 3, R4, R5). The new transfer function is obtained in 3C magnifying factor (in brackets) when compared to 3, while C, R4 and R5 are kept fixed: solid state switches were applied for this task. R4/R5 can be as high as needed, e.g., 103, to avoid large capacitors with high leakage currents, i.e., electrolytic:2 input bias current and input offset voltage become responsible for the output DC offset at IC1: a small non-zero DC voltage appears at the R3-R4 node. As R4-R5 acts as a voltage divider, the DC voltage is amplified to reach the output DC offset at IC1. Thus, R5 ≪ R3 should be chosen to minimize and achieve a constant DC voltage at the R3-R5 node. The tests showed that output DC offset at IC1 remained constant, notwithstanding the R3C values selected.At this point, ICTherefore, the stage shown in These characteristics will also be featured by the second implementation, shown in 4 is equal to R1 to create a voltage adder with the feedback-loop. The R3C magnifying factor in 5/R6, compared to 3. The magnifying factor allows low cut-off frequencies, avoiding large capacitors with leakages and permitting R3 standard values.RBoth designs add resistors or an op-amp stage in the feedback-loop to create the magnifying factor, if compared with 2.3.B. The critical path in the design in 5-R6 op-amp inverting stage and the resistor R3. The loop op-amps IB contribute to the spurious DC levels at vo and the main contribution comes from IC2. Thus, the output DC voltage Vo,dc can be constrained with low IB op-amps. On the other hand, the op-amp input offset voltage VOS at IC1 is transferred to vo by the gain resistors R1 and R2, but this DC level is compensated by the feedback network. However, the VOS at IC2 cannot be neglected. Thus, the VOS at IC2 appears at the R6-R3 node and it is transferred to the vo node with the magnifying factor R5/R6. Hence, Vo,dc depends both on IB and VOS and is calculated by Spurious output DC levels depend on the closed-loop behaviour and the op-amp input bias currents IB can be highly dependent on the temperature and responsible for the thermal output voltage drifts. Thus, a thermal increment in the op-amp IB will be multiplied and translated into an increment in the output DC voltage Vo,dc, as described by I3-R4-R5 resistors. Here, both IB and VOS of the loop op-amp IC2 are responsible for the spurious DC levels Vo,dc. As in the previous design, IB and VOS effects are transferred to vo through the R3-R4-R5 network as described by B can be highly dependent on the temperature and, hence, responsible for the thermal voltage drifts. Later on, numeric values for the thermal drifts will be given for a set of six op-amps in a trial:The critical path in the design depicted in 2.4.ia node and ending at vhpf node. The stage in ia starting node and the vhpf ending node. Two transient suppressors are included to protect the inputs from electrostatic discharges.A bioelectric amplifier stage was designed in order to evaluate the performance of the circuits in 2 and R3 values. Then, gain and cut-off frequency can be selected by combining both switches. Each switch has three input bits for way selection, which allows for eight resistor values. Hence, there are 64 possible combinations, i.e., eight different cut-off frequencies for each of the eight selectable gains. The minimum cut-off frequency in trial was 0.016 Hz and the maximum 31.83 Hz. The switching procedure may be commanded with a digital controller.Both implementations, hpf node. It trims the constant output DC offset, and accommodates the DC-level to the MAX7401 low-pass filter (LPF) subsequent stage. The LPF stage starts at the vshft node and is composed of an RC anti-aliasing filter and the MAX7401 switched capacitor filter (SCF).2.5.nw in the white region and the corner frequency fc, which is the 1/f noise boundary, are necessary and supplied by the manufacturer datasheet. Hence, the voltage noise spectral density en was considered as the composition of the 1/f low-frequency flicker noise and the white noise, as shown in The bioelectric amplifier noise is analysed with the test circuit depicted in n,rms by composing the 1/f noise and the white noise: the passband limits are defined by the lower cut-off frequency fHPF determined by the HPF, and the upper cut-off frequency fLPF. From the test-circuit shown in LPF cut-off frequency is determined by the IA and the SCF low-pass combined response.hpf node is calculated with n,HPF in n,opamp.From the test circuit in In order to compare the rms voltages with practical measurements on an oscilloscope, it is useful to convert the rms noise values into peak-to-peak values. Thus, a 6.6 conversion factor was applied in hpf and vo but it was a minor contribution compared to the IA and HPF gain stages contribution. In fact, the oscilloscope noise measure at vo acts as a reference to compare to, as it will exceed the IA and HPF contribution.The calculated results were compared with the noise measured with an oscilloscope at the output of the test circuit in 3.3.1.o, shown in T = 50·G, as indicated in As in standard filters, the HPF capacitor can require deblocking for low cut-off frequencies at the initial setup. The electronic switching control system and the small capacitance C allow this operation. In order to make it work, the highest HPF frequency is selected once the electrodes are attached to the subject: the HPF delay constant is low enough to rapidly discharge the capacitor. Afterwards, the required cut-off frequency is automatically established by the control system.− input and the IA v+ input was grounded. Then, −2.52 V were obtained at the via input node. The achieved output offset was 3.48 V at the vhpf node for any gain or HPF frequency resistor configuration.The output offset of the stage in 2 in 3 was fixed to 1 MΩ. As before, an INA114 instrumentation amplifier was connected at the input of the stage in i.e., 0.05 Vdc at the IA v− input, IA v+ grounded and, hence, −2.52 V were achieved at the input node vi in The circuit in 3 causes the deviations. This current depends on the manufacturer, the capacitance and the electrolytic capacitor specifications. In comparison, the designs in The most remarkable conclusion regarding the results in 3.2.o. However, the underlying technology of the op-amps involved will affect this value. Both designs are configuration-selectable (gain and cut-off frequency), so it is expected that the offset values will be configuration-dependant. Hence, two parameters were measured: output DC offset Vhpf-OS, and the maximum variation of the output DC offset, ΔVhpf-OS, for all possible configurations.Both circuits in OS supplied by the manufacturer, among other specifications. The reference configuration to measure Vhpf-OS is the combination of the lowest gain and the highest HPF cut-off frequency. The circuit performance in hpf node in the test circuit in hpf node, after replacing the HPF circuit in Six op-amps were analysed. hpf node. The values were obtained from OS and IB manufacturer specifications in hpf-OS is the combination of the lowest gain and the highest HPF cut-off frequency. The statistical relevance of the offset values relies on these specifications. Thus, the results shown in 3.3.hpf-OS was measured at the laboratory. Tests were performed with a set of op-amps from different manufacturers and a temperature sensor attached to the op-amp package. Wide excursion values were measured to confirm the Vhpf-OS variation with the temperature. However, the sensor fixture was not reliable enough to give accurate drift values. Therefore, in order to tabulate the drifts, the thermal deviations were obtained from the op-amp IB drift with temperature supplied by the manufacturer. While the circuit is intended to work indoors, at room temperature, an increase in the package temperature is expected during the normal operation. Hence, current and voltage drifts refer to 25 °C in this analysis. Thus, B increment (ΔIB) when the op-amp temperature is increased from 25 °C to 50 °C, 75 °C and 100 °C.Thermal stability of the output offset VT,hpf-OS in B with the ΔIB values in 1 = 1 kΩ, R2 ranges from 47 kΩ to 2 MΩ, R3 is comprised between 10 kΩ and 470 kΩ, R4 = 100 kΩ and R5 = 100 Ω. Thus, the resulting expression in 3 value, i.e., 10 kΩ. The results in 3 value selected:Output voltage drift with temperature ΔVB. Later on, a set of 6 specific op-amps from different manufacturers were analysed in order to compare the thermal performance with the proposed HPF designs.3 value, the worse the drift obtained. So, in order to compare the stabilities, 3 value in trial.The thermal drifts were also analysed for the set of 6 op-amps previously selected. As expected, the higher the R3.4.hpf. As explained previously, the noise calculations are valid for both circuits in Noise performance was calculated from the manufacturer specifications. Afterwards, the calculations were compared with the measurements obtained from the oscilloscope. o of the test circuit in + and v− inputs were grounded. The configuration set shown in Noise was measured at the output vAs previously done, the noise analysis was also performed for the set of six op-amps in trial. Thus, the results in nw = 5.9 nV/√Hz at 1 kHz and fc = 30 Hz.The whole set of six op-amps was measured with the oscilloscope to evaluate noise performance. To avoid excessive data, the lowest noise op-amp tests are displayed in In order to give statistical relevance to the noise measurements in As an example, 4.The frequency response was consistent with the calculated HPF cut-off frequencies. 4.1.i.e., the IA. The IA input noise is transmitted to the HPF input, amplified by GIA = 50. Hence, the IA maximum noise contribution at the HPF output is GIA·GHPF,max·en,IA = 105·en,IA, while the HPF contribution at the HPF output is GHPF,max·en,HPF = 2·103·en,HPF. Hence, according to the values in As expected, most of the noise contribution in the circuit in trial comes from the front stage, hpf node) for the TL084 op-amp and the set of six configurations described in i.e., the IA.From the time base in 4.2.The HPF output is expected to deliver signals within a dynamic range of several volts, e.g., ±5 V or ±15 V. This range is closely related to the HPF power supply. The ulterior stages and the ADC input dynamic range are the references against which to compare the HPF output DC offset. We can take a 5 V dynamic range for the ADC as a reference for these analyses.i.e., VOS and IB. FET op-amps give the lowest IB. However, some non-FET precision op-amps can deliver very low VOS values, such as the MAX44252 or the OPA4277. hpf-OS and ΔVhpf-OS.The HPF output DC offset depends on the op-amp technology, and hpf-OS represents the baseline output DC offset and it can be compared with the ADC dynamic range. The highest Vhpf-OS is for the TL084 and hardware trimming would be required. The test circuit in hpf-OS. The lowest Vhpf-OS is for the MAX44252 or the AD8513, depending on the configuration.Vhpf-OS adds valuable information, as the circuit is intended to work as a PGA. Hence, it would be desirable to have a minimum ΔVhpf-OS within the set of available gains. According to i.e., the OPA4277 and the MAX44252.However, ΔVB is the main responsible for the ΔVhpf-OS values. The designs in 3 to switch the cut-off frequency, as shown in the test circuit in 2 and keeping R3 constant results in ΔVhpf-OS = 0 for any op-amp. On the other hand, the value Vhpf-OS is related to a combination of VOS and IB, and depends on the configuration, i.e., the circuits in From hpf-OS and ΔVhpf-OS. Low frequency 1/f noise could be a drawback with FET op-amps. However, from the previous noise analyses, it was concluded that the front stage noise, from the IA, is the main contribution to the noise budget. Hence, the best results in terms of Vhpf-OS and ΔVhpf-OS are achieved with the AD8513 and the OPA4132. According to B and moderate VOS. Nevertheless, a general purpose TL084 FET op-amp can also deliver an excellent ΔVhpf-OS performance when the Vhpf-OS is trimmed at a subsequent stage. On the other hand, it is worth to highlight that the Vhpf-OS is not related to the gain G, as seen on 1 or R2. This fact was also verified in the laboratory.Thus, the performance depends on the stage and the op-amp selected. As a trade-off, it can be concluded that the FET op-amps deliver the best results in terms of Vhpf-OS,typ values and within the Vhpf-OS,max interval of values. The same conclusion is applied to ΔVhpf-OS,typ and ΔVhpf-OS,max. Altough the ΔVhpf-OS = 5 mV measured for the AD8643 was higher than the calculated values (about 0.47 mV), it is considered a measurement error due to a lack of precision. The rest of the ΔVhpf-OS measured values in The data in about 0.4 mV, it i4.3.T,hpf-OS = 3,260 mV. Also, the OPA4132 gives a high ΔVT,hpf-OS = 1,868 mV. Hence, for low power applications at room temperature (25 °C), when the thermal drifts are constrained, a general purpose op-amp could be chosen. But depending on the application, the environmental conditions and the cooling of the enclosure, a careful selection of the op-amp is required. Hence, the op-amp choice, in terms of DC offset, involves a trade-off between thermal stability and output DC offset.5.DC analysis is not often described in depth in bioelectric amplifiers. However, it cannot be neglected, as high gains and low cut-off frequencies pose certain problems. This paper proposes a simple intermediate stage to overcome the DC issues. Hence, two new designs were analysed and tested to implement this stage. They feature: (i) DC coupling; (ii) selectable gain (PGA) and HPF; (iii) low and constant output DC offset for any configuration; (iv) very low HPF cut-off frequencies, avoiding large capacitors; and (v) capacitor deblocking strategy.hpf-OS are achieved when switching gains and cut-off frequencies, but output DC offset trimming can be required in order to minimize the constant offset Vhpf-OS at the output. Precision FET op-amps with low IB and low VOS avoid this trimming. However, variable thermal conditions can involve a trade-off between thermal stability and output DC offset when selecting the op-amp. On the other hand, the 1/f noise can be relevant in FET op-amps, although it is not a drawback as the IA front-end noise is the main contribution to the noise budget. For switching purposes, it is worth noting that Vhpf-OS is not dependant on the stage gain G.From the analyses, it is concluded that general purpose FET op-amps can be used in room temperature conditions: small offset variations ΔV5. Thus, this IA-PGA reduced set can deliver the selectable gain and high pass filtering required for a bioelectric amplifier.Hence, this single PGA stage can reduce the DC coupling problems with minimum or no trimming. It can be combined with a monolithic IA, or an adapted differential front-end with moderate gain. The test circuit was designed with a set of HPF cut-off frequencies ranging from 0.016 Hz to 31.83 Hz and a set of gains ranging from 2,350 to 10